Herpes simplex virus 1 (HSV-1) is a ubiquitous viral pathogen that, while rarely lethal, nevertheless has the ability to cause severe morbidity in a subset of patients. Among the more serious potential consequences of HSV-1 infection is the development of HSV-1 keratitis, the most common cause of infectious blindness in the USA, with ~35,000 new cases each year. Currently available drugs do not effectively treat HSV-1 keratitis, and a new treatment modality is therefore clearly needed. In this grant application, we propose to optimize the bacterial CRISPR/Cas mechanism of adaptive antiviral immunity in order to efficiently cleave and inactivate latent HSV-1 genomes in neuronal cells in vivo. Our initial goal is to develop vectors based on adeno-associated virus (AAV), which has been widely used for gene therapy in humans, to express the novel smaller Cas9 protein recently identified in Staphylococcus aureus (Sau), together with small guide RNAs (sgRNAs) specific for HSV-1. After optimization and demonstration of function in cultured cells, these AAV vectors will be introduced into mice containing trigeminal ganglia latently infected by HSV-1 and the potential reduction in viral DNA load and loss of reactivation potential assayed. In this way, we hope to provide proof-of-principle for the idea that CRISPR/Cas systems can be repurposed as a novel tool for the effective elimination of latent HSV-1 DNA genomes in vivo.